1. Technical Field
This invention relates generally to fluorescence polarization immunoassays and reagents useful therein, and particularly to such an assay for amphetamine and methamphetamine. The invention provides a preincubation step which is effective to eliminate cross reactivity to .beta.-hydroxyamines.
2. Background Art
Amphetamine and methamphetamine are sympathomimetic phenethylamine derivatives having central nervous system stimulant activity. These drugs have been used for the treatment of obesity, narcolepsy, and hypotension. Because of their stimulant effects, the drugs are commonly sold illicitly and abused. Physiological symptoms often associated with very high amounts of ingested amphetamine and methamphetamine include elevated blood pressure, dilated pupils, hyperthermia, convulsions, and acute amphetamine psychosis.
The biological fluid tested most frequently is urine. Other biological fluids have no been extensively investigated for use in assays for the detection of amphetamine and methamphetamine. In the past, amphetamines have been detected by a number of techniques, including thin-layer chromatography (TLC), gas chromatography (GC), and high performance liquid chromatography (HPLC). These methods generally involve chemical extractions of the drugs, complicated procedures requiring highly trained personnel and lengthy assay times.
In general, competitive binding immunoassays have provided a preferable alternative to chemical methods such as GC and HPLC. Typically, competitive binding immunoassays are used for measuring ligands in a test sample. Generally, a "ligand" is a substance of biological interest to be determined quantitatively by a competitive binding immunoassay technique. The ligands compete with a labeled reagent, or "ligand analog," or "tracer," for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of ligand in the sample determines the amount of ligand analog which binds to the antibody: the amount of ligand analog that will bind-is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
An accurate and reliable immunoassay requires that antibody "cross-reactivity" (recognition of compounds other than the desired ligand or ligands) be minimized. In the case of assays for amphetamine and methamphetamine it is known that derivatives of .beta.-phenethylamine, such as .beta.-hydroxyphenethylamine compounds, may be strong interferants. One such .beta.-hydroxyphenethylamine, the drug phenylpropanolamine, is found frequently in decongestants sold over the counter. U.S. Pat. No. 3,856,469 discloses removal of .beta.-hydroxyphenethylamine interference from a sample intended for amphetamine or methamphetamine analysis by treating the sample at a pH greater than 8.0 with an amount of aqueous periodate in the presence of ammonium hydroxide. In addition to requiring sample treatment at a basic pH, the aqueous pretreatment in U.S. Pat. No. 3,856,469 is suggested as useful only preceeding sample evaluation by thin layer chromatography and immunoassays by radioimmunoassay, electron spin resonance technique or enzyme technique.
Fluorescence polarization provides an alternative quantitative or qualitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody conjugate having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane-polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate and an excited population of molecules is randomized much more quickly. As a result, the light emitted from the unbound tracer molecules is depolarized.
To date no fluorescence polarization assay for determining amphetamine and/or methamphetamine in a single assay has been disclosed.
Accordingly, a need exists for providing a method and reagents for performing a reliable and accurate fluorescence polarization assay for both amphetamine and methamphetamine in biological fluids such as urine. A further need exists for conducting aqueous periodate pretreatment of urine samples to be tested for amphetamine and/or methamphetamine without the addition of pH raising constituents, such as bases.